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PMID: 8458958 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

Journal of clinical microbiology ·Vol. 31 ·No. 3 ·1993-03-00 ·Pages 646-52

Meier A, Persing DH, Finken M, Böttger EC

Abstract

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.

MeSH Terms
Bacteria/genetics,isolation & purification,pathogenicity Base Sequence DNA Damage/radiation effects DNA, Bacterial/genetics,isolation & purification DNA, Ribosomal/genetics Drug Contamination Humans Indicators and Reagents/isolation & purification,radiation effects Methoxsalen Molecular Sequence Data Mycobacterium tuberculosis/genetics,isolation & purification Osteomyelitis/microbiology Polymerase Chain Reaction/methods RNA, Ribosomal, 16S/genetics Ultraviolet Rays
Chemicals
DNA, Bacterial DNA, Ribosomal Indicators and Reagents RNA, Ribosomal, 16S Methoxsalen
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Meier A
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Persing D H
Finken M
Böttger E C
References (43)
43 references, click to expand
  1. Probe directed at a segment of Rickettsia rickettsii rRNA amplified with polymerase chain reaction.
    J Clin Microbiol. 1989 Dec;27(12):2692-6 PMID: 2512323
  2. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction.
    J Clin Microbiol. 1990 Sep;28(9):1942-6 PMID: 2095137
  3. Use of psoralen as extinguisher of contaminated DNA in PCR.
    Nucleic Acids Res. 1990 Nov 25;18(22):6739 PMID: 2251160
  4. 16S ribosomal DNA amplification for phylogenetic study.
    J Bacteriol. 1991 Jan;173(2):697-703 PMID: 1987160
  5. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium.
    Int J Syst Bacteriol. 1990 Oct;40(4):323-30 PMID: 2275850
  6. Direct detection and amplification of Helicobacter pylori ribosomal 16S gene segments from gastric endoscopic biopsies.
    Diagn Microbiol Infect Dis. 1990 Nov-Dec;13(6):473-9 PMID: 1703940
  7. Differentiation of Mycobacterium species by direct sequencing of amplified DNA.
    J Gen Microbiol. 1990 Sep;136(9):1915-20 PMID: 2283506
  8. Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction.
    Nucleic Acids Res. 1991 Jan 11;19(1):99-107 PMID: 2011516
  9. Taq polymerase contains bacterial DNA of unknown origin.
    Mol Cell Probes. 1990 Dec;4(6):445-50 PMID: 2087233
  10. DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia.
    Lancet. 1991 Jun 8;337(8754):1378-9 PMID: 1674765
  11. Bacterial evolution.
    Microbiol Rev. 1987 Jun;51(2):221-71 PMID: 2439888
  12. Synthetic oligonucleotide probes complementary to rRNA for group- and species-specific detection of mycoplasmas.
    Isr J Med Sci. 1987 Jun;23(6):742-6 PMID: 2889700
  13. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.
    Science. 1989 Mar 10;243(4896):1360-3 PMID: 2466341
  14. Broad range DNA probes for detecting and amplifying eubacterial nucleic acids.
    FEMS Microbiol Lett. 1989 Jan 1;48(1):19-24 PMID: 2469620
  15. Avoiding false positives with PCR.
    Nature. 1989 May 18;339(6221):237-8 PMID: 2716852
  16. Rapid, sensitive diagnosis of malaria based on ribosomal RNA.
    Lancet. 1989 Jun 17;1(8651):1343-6 PMID: 2567370
  17. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.
    Nucleic Acids Res. 1989 Jul 11;17(13):4937-46 PMID: 2668874
  18. Rapid identification of Campylobacter species using oligonucleotide probes to 16S ribosomal RNA.
    Mol Cell Probes. 1989 Jun;3(2):133-42 PMID: 2770753
  19. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.
    Nucleic Acids Res. 1989 Oct 11;17(19):7843-53 PMID: 2798131
  20. Use of UV irradiation to reduce false positivity in polymerase chain reaction.
    Biotechniques. 1991 Apr;10(4):442, 444, 446 PMID: 1867851
  21. Phylogeny of the Whipple's-disease-associated bacterium.
    Lancet. 1991 Aug 24;338(8765):474-5 PMID: 1714530
  22. Detection and identification of mycoplasmas by amplification of rDNA.
    FEMS Microbiol Lett. 1991 Jun 1;65(1):37-42 PMID: 1874400
  23. The 16S rRNA nucleotide sequence of Mycobacterium leprae: phylogenetic position and development of DNA probes.
    FEMS Microbiol Lett. 1991 May 15;64(2-3):231-7 PMID: 1884981
  24. Polymerase chain reaction: trenches to benches.
    J Clin Microbiol. 1991 Jul;29(7):1281-5 PMID: 1885726
  25. Detection of DNA contamination in Taq polymerase.
    Biotechniques. 1991 Aug;11(2):176-7 PMID: 1931012
  26. Microheterogeneity within rRNA of Mycobacterium gordonae.
    J Clin Microbiol. 1992 Apr;30(4):1049-50 PMID: 1374075
  27. Detection of the etiologic agent of human ehrlichiosis by polymerase chain reaction.
    J Clin Microbiol. 1992 Apr;30(4):775-80 PMID: 1374076
  28. Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene.
    J Clin Microbiol. 1992 Apr;30(4):796-800 PMID: 1374077
  29. Species-specific assessment of Mycobacterium leprae in skin biopsies by in situ hybridization and polymerase chain reaction.
    Lab Invest. 1992 May;66(5):618-23 PMID: 1573855
  30. Disseminated "Mycobacterium genavense" infection in patients with AIDS.
    Lancet. 1992 Jul 11;340(8811):76-80 PMID: 1352014
  31. Identification of the uncultured bacillus of Whipple's disease.
    N Engl J Med. 1992 Jul 30;327(5):293-301 PMID: 1377787
  32. Shedding light on PCR contamination.
    Nature. 1990 Jan 4;343(6253):27 PMID: 2296286
  33. Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA.
    FEMS Microbiol Lett. 1989 Nov;53(1-2):171-6 PMID: 2482222
  34. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology.
    J Bacteriol. 1990 Feb;172(2):762-70 PMID: 1688842
  35. Specific Neisseria gonorrhoeae DNA-probes derived from ribosomal RNA.
    J Gen Microbiol. 1989 Jun;135(6):1735-45 PMID: 2482333
  36. Rapid and simple method for purification of nucleic acids.
    J Clin Microbiol. 1990 Mar;28(3):495-503 PMID: 1691208
  37. Frequent contamination of Taq polymerase with DNA.
    Clin Chem. 1990 Jun;36(6):1258-9 PMID: 2357811
  38. A general method to generate DNA probes for microorganisms.
    Biotechnology (N Y). 1990 Mar;8(3):233-6 PMID: 1369980
  39. Detection and identification of mycobacteria by amplification of rRNA.
    J Clin Microbiol. 1990 Aug;28(8):1751-9 PMID: 2203812
  40. Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dammini ticks.
    Science. 1990 Sep 21;249(4975):1420-3 PMID: 2402635
  41. Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level.
    FEMS Microbiol Lett. 1990 Jul;58(2):197-203 PMID: 2275731
  42. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.
    Gene. 1990 Sep 1;93(1):125-8 PMID: 2227421
  43. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens.
    N Engl J Med. 1990 Dec 6;323(23):1573-80 PMID: 2233945
Article Info
Journal
Journal of clinical microbiology
Abbr.
J Clin Microbiol
ISSN
0095-1137
DOI
10.1128/jcm.31.3.646-652.1993
Published
1993-03-00
Pages
646-52
Language
English
Region
United States
NLM ID
7505564
PMCID
PMC262835
Subset
IM
External Links
PubMed source DOI: 10.1128/jcm.31.3.646-652.1993 PMC Full Text View journal details
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